Development and Characterization of Potent Succinate Receptor Fluorescent Tracers

The succinate receptor (SUCNR1) has emerged as a potential target for the treatment of various metabolic and inflammatory diseases, including hypertension, inflammatory bowel disease, and rheumatoid arthritis. While several ligands for this receptor have been reported, species differences in pharmacology between human and rodent orthologs have limited the validation of SUCNR1’s therapeutic potential. Here, we describe the development of the first potent fluorescent tool compounds for SUCNR1 and use these to define key differences in ligand binding to human and mouse SUCNR1. Starting from known agonist scaffolds, we developed a potent agonist tracer, TUG-2384 (22), with affinity for both human and mouse SUCNR1. In addition, we developed a novel antagonist tracer, TUG-2465 (46), which displayed high affinity for human SUCNR1. Using 46 we demonstrate that three humanizing mutations on mouse SUCNR1, N181.31E, K2697.32N, and G84EL1W, are sufficient to restore high-affinity binding of SUCNR1 antagonists to the mouse receptor ortholog.


■ INTRODUCTION
Long viewed primarily as a metabolic intermediate of the tricarboxylic acid cycle, succinate has recently been attributed novel physiological roles beyond cellular energy production. In 2004, succinate was reported as the endogenous ligand for GPR91, subsequently renamed the succinate receptor (SUCNR1), 1,2 a G protein-coupled receptor mainly signaling through a Gα i -mediated pathway. 3−7 The receptor is expressed in a wide range of cells and tissues, including kidney, 1 liver, 8 adipose, 9 heart, 10 retina, 11 and immune cells. 12,13 SUCNR1 has been implicated in a range of pathological conditions such as hypertension, 14−16 liver fibrosis, 17,18 rheumatoid arthritis, 19 agerelated macular degeneration, 20 cancer, 21,22 and periodontitis. 23 Many studies suggest a pro-inflammatory role of the succinateinduced stimulation of SUCNR1. For example, the succinate-SUCNR1 axis drives the Toll-like receptor-induced inflammatory cytokine production in dendritic cells, 12 while SUCNR1 activation in pro-inflammatory macrophages results in increased production of IL-1β cytokines. 19 In contrast, several other studies indicate an anti-inflammatory role of SUCNR1 stimulation in myeloid cells 24 and neural stem cells. 25 Although there is a growing body of evidence suggesting that inhibition of SUCNR1 is of therapeutic interest, the conflicting roles of the receptor in inflammation leave the space open for therapeutic development of both agonists and antagonists.
A number of synthetic SUCNR1 modulators have been reported, including a series of naphthyridines, 26 non-metabolite partial agonists with nanomolar potency at mouse (m) and human (h) SUCNR1, 2 and a nanomolar potency human-specific antagonist NF-56-EJ40 (1). 27 The lack of high-quality tool compounds and in particular antagonists with activity on rodent receptor orthologs is the major obstacle for in vivo studies to validate the therapeutic potential of SUCNR1.
Fluorescently labeled ligands have proven to be valuable pharmacological tools for investigations of ligand binding to GPCRs. 28 The fluorescent tracers possess many advantages over conventional radiolabeled tracers, including practical convenience concerning safety, waste disposal, and cost. Moreover, fluorescence-based binding assays enable studies of real-time binding and visualization of ligand−receptor complexes in intact cells and allow for non-wash homogeneous HTS-compatible assay formats when employed with resonance energy transfer (RET)-based techniques. 28−31 Nano bioluminescence resonance energy transfer (NanoBRET) assays have been successfully used to investigate binding of ligands targeting other carboxylate-sensing receptors, including the free fatty acid receptors, FFA1 and FFA2. The solvatochromic dye 4-amino-7nitrobenzoxadiazole (NBD) has been employed in these FFA1 and FFA2 tracers, 32,33 and although it has low brightness and fluorophores emitting in the green/yellow sometimes present challenges as tracers due to cellular autofluorescence, NBD has an absorption band that overlaps almost perfectly with the Nanoluciferase (Nluc) emission band, is relative easy to introduce at primary amines, and has a good chemical stability. Therefore, we aimed to develop potent NBD-based fluorescent agonist and antagonist tracers for SUCNR1 to be used in a NanoBRET assay 34 based on current SAR insight for SUCNR1 agonists TUG-1689 (2) and TUG-1688 (3) 2 and the recent  (Figure 1). We then aimed to use these tracers to define the basis for the lack of binding affinity for 1 and other SUCNR1 antagonists at the mouse ortholog of SUCNR1.

■ RESULTS AND DISCUSSION
The agonist tracer precursors were synthesized from intermediate 4 2 or by a fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH)-mediated amide coupling between 6-chloropicolinic acid (10) and the dimethyl ester of L-aspartic acid (11) (Scheme 1). 35 The amide intermediate 12 underwent Suzuki coupling with parahydroxyphenylboronic acid using the fourth-generation XPhos precatalyst, and the phenol was alkylated with various Bocprotected aminoalkyl halides and tosylates. The esters were hydrolyzed under basic conditions to give the Boc-protected tracer precursors, while the free amine tracer precursors  underwent HCl-mediated Boc-deprotection before hydrolysis. The first NBD tracer 7 was synthesized from the Bocdeprotected methyl ester intermediate 6 by substitution with NBD-Cl followed by ester hydrolysis. The extended amine linker analogue 17 was synthesized from the phenol intermediate 4 by first an alkylation with 1,6dibromohexane followed by substitution with tert-butyl (2aminoethyl)carbamate and finally hydrolysis of the esters (Scheme 2). 4 was alkylated with 6-bromohexan-1-ol followed by oxidation of the hydroxy intermediate (18) to the corresponding aldehyde 19 that was subjected to reductive aminations with methyl-and dimethylamine hydrochlorides followed by base-promoted ester hydrolyses to give Nmethylated and N,N-dimethylated analogues 20 and 21.
To get NBD attached selectively at the primary amine, the tracer was synthesized from aldehyde 19 that underwent reductive amination with NBD coupled to diaminoethyl, and hydrolysis of the esters gave the tracer 22.
The antagonists were synthesized following the same overall synthetic strategy starting from the methyl ester of 2-(2aminophenyl)acetic acid (23) that underwent HATU-mediated amide coupling with halogen-substituted benzoic acid derivatives (Scheme 3). These amide intermediates were converted to various biphenyl intermediates by Suzuki coupling either directly using boronic acids or by a two-step, one-pot protocol forming first the boronic ester in situ from the first aryl halide followed by Suzuki coupling to a second aryl halide. The parachloro analogues were directly hydrolyzed to give the final antagonists. The alkoxy analogues were prepared from the phenol intermediates 36 and 37, first by alkylation with various Boc-protected aminoalkyl halide and tosylates, followed by basic ester hydrolysis, and finally Boc-deprotection using TFA. The piperazine analogues were hydrolyzed after the Suzuki coupling, and 46 was prepared from 45 by a Boc-deprotection followed by a substitution reaction with NBD-Cl.
First, we set out to investigate five different linkers on the mouse agonist scaffold 2 (Table 1). Simple aliphatic linkers were chosen to investigate the optimal linker length, including one PEG-based linker to examine effects of polarity, all containing a Boc-protected amine as a stable surrogate for the fluorophore that to some degree mimics the steric bulk but at the same time is a handle for easy introduction of the NBD-fluorophore after deprotection. Unfortunately, all linkers seemed to reduce the potency on human and mouse SUCNR1, with the C5-linker analogue 8c being slightly better tolerated, showing only a 2.5fold decrease in potency on mSUCNR1. Exploring the same five linkers on the agonist with selectivity for hSUCNR1 (3) led to a dramatic decrease in potency for all compounds (15a−e) on hSUCNR1 while only affecting the potency slightly on mSUCNR1. Based on this, we selected 8c as the best tracer precursor and converted the compound to the corresponding NBD-tracer 7, which fully sustained the potency on mSUCNR1 (pEC 50 = 6.70 ± 0.16) but showed poor potency on hSUCNR1 (pEC 50 = 5.84 ± 0.31).
Despite its relatively low potency for hSUCNR1, we next tested whether 7 may still be a useful agonist fluorescent tracer for either human or mouse SUCNR1 in BRET-based binding assays. For this assay, versions of both human and mouse SUCNR1 were tagged at their extracellular N termini with Nluc and expressed in Flp-In T-REx 293 cells. To confirm that these Nluc-modified receptors did not have altered pharmacology, we first compared the expression level of the Nluc-hSUCNR1 and Nluc-mSUCNR1 constructs using their Nluc emission, finding no statistical difference in expression levels between the two orthologs (mSUCNR1 expression was 85 ± 10% of hSUCNR1 expression). We next demonstrated that succinate activated both Nluc-mSUCNR1 (pEC 50 = 5.69 ± 0.11) and Nluc-hSUCNR1 (pEC 50 = 4.80 ± 0.01) in a cAMP assay with comparable potency to what we observed previously using the unmodified receptors. Likewise, 7 showed high potency at Nluc-mSUCNR1 (pEC 50 = 7.07 ± 0.07) but significantly reduced potency at Nluc-hSUCNR1 (pEC 50 = 5.94 ± 0.18), in excellent alignment with our initial results for 7 using unmodified SUCNR1 constructs. When tested in saturation BRET binding assays, 7 demonstrated clear specific binding to both hSUCNR1 ( Figure 2A, Figure S1A) and mSUCNR1 ( Figure 2B, Figure  S1B). Surprisingly, although the affinity of 7 for mSUCNR1 (K d = 1.16 μM) was higher than it was for hSUCNR1 (K d = 1.58 μM), this difference was not significant and was less pronounced than might have been expected based on the 7−13-fold higher  potency this compound displays for mSUCNR1 over hSUCNR1 in cAMP assays. Interestingly, the measured affinity of 7 for hSUCNR1 matches very well with the potency of this compound in the cAMP assay at the human receptor, suggesting that for hSUCNR1 there is no receptor reserve operating in 7 cAMP signaling. Together, these results suggest that, despite having similar affinities at hSUCNR1 and mSUCNR1, the greater potency of this compound for mSUCNR1 is likely derived from differences in coupling efficiency of human and mouse SUCNR1 for the cAMP pathway.
Considering the relatively poor potency for 7 at hSUCNR1, we next used computational modeling to examine the potential binding mode of the agonist tracer precursors. Knowing that the antagonist 1 can displace the agonist tracer 7 and that the  Saturation binding data are shown as mean ± SEM from three independent experiments carried out in duplicate. Saturation binding data were globally fit to a total and non-specific binding equation, yielding a K d value of 0.99 μM (95% CI = 0.45−2.15 μM) for hSUCNR1 and 1.17 μM (95% CI = 0.90−1.51 μM) for mSUCNR1. Kinetic BRET binding experiments are shown for Nluc-hSUCNR1 (C) and Nluc-mSUCNR1 (D). The kinetic data are shown as specific BRET, subtracting signal obtained when treating with equivalent concentrations of 22 in the presence of 100 μM 17. Kinetic binding data are shown as mean ± SEM from three independent experiments carried out in triplicate and were globally fit to an equation for binding of multiple concentrations of labeled ligand. compounds share many common structural features, including an acidic headgroup connected by an amide to a biphenyl core, we used the X-ray structure of 1 in complex with the humanized rSUCNR1 27 as template and used induced-fit docking to better compensate for structural differences between the active and inactive states of the receptor. Based on these results, we hypothesized that a positive charge in the linker could gain additional ionic interactions with Glu18 in hSUCNR1 and Glu14 conserved in human and mouse SUCNR1 and thereby increase affinity (Figure 3). To investigate this, we Bocdeprotected all the furane-based tracer precursors with alkylene linkers and tested the tracer precursors with free amines (9a−d, Table 2). To our satisfaction, all compounds increased in potency on both receptor orthologs, with a significant preference for the longer 9d for hSUCNR1 and marginally increased potency on mSUCNR1.
To avoid introduction of a second stereocenter in the ligands, we aimed at extending the linker at the terminal amine to thereby have a positive charge as either a secondary or tertiary amine rather than incorporating the primary amine on the linker. To make sure this was a viable strategy, we first made the corresponding N-methyl (20) and N,N-dimethyl (21) analogues. Both compounds showed preserved potency on mSUCNR1 and a small but acceptable decrease on hSUNCR1. We therefore decided to extend the secondary amine further to accommodate the NHBoc handle for introduction of the NBDfluorophore. The tracer precursor 17 nicely regained some of the lost potency on hSUCNR1 and showed nanomolar potency on both receptor orthologs (pEC 50 = 6.97−7.40). Thus, the corresponding NBD-tracer 22 was synthesized, and although the potency decreased slightly on both orthologs (pEC 50 = 6.84−7.16), the compound represents a SUCNR1 tracer with improved potency for both the hSUCNR1 and mSUCNR1.
Before testing 22 in BRET binding assays, we first confirmed that its activity was retained at Nluc-tagged hSUCNR1 (pEC 50 = 7.38 ± 0.08) and mSUCNR1 (pEC 50 = 7.41 ± 0.06). Testing 22 in saturation BRET binding assays demonstrated that this compound displays specific binding to both hSUCNR1 ( Figure  4A, Figure S2A) and mSUCNR1 ( Figure 4B, Figure S2B), with K d values of 990 and 1170 nM, respectively. These results show that 22 has comparable affinity to our first tracer, 7, at mSUCNR1, but with a modest improvement in affinity for hSUCNR1. However, improvement in affinity when comparing 22 to 7 was much less pronounced (1.6-fold) than the improvement in cAMP potency (10−14-fold) between the compounds, perhaps suggesting that the amine in 22 has a greater effect on signal transduction efficiency than it has on binding affinity.
We next aimed to assess whether 22 could be used in real-time binding kinetic assays. Experiments were conducted by measuring the association binding of multiple concentrations of 22 and globally fitting these data to a single equation in order to provide estimates of both on (k on ) and off (k off ) rates, as well as the dissociation constant (K d ). Assessment of the binding kinetics of 22 at hSUCNR1 resulted in a k on of 23100 M −1 min −1 (95% CI = 22000−24000 M −1 min −1 ) and k off of 0.014 min −1 (95% CI = 0.008−0.021 min −1 ) ( Figure 4C). Deriving K d from these kinetic studies yields a value of 620 nM, in good alignment with the affinity measured for 22 at hSUCNR1 in the saturation binding assay ( Figure 4A). Kinetic analysis of 22 binding to mSUCNR1 yielded a k on of 85800 M −1 min −1 (95% CI = 84000−88000 M −1 min −1 ) and k off of 0.13 min −1 (95% CI = 0.12−0.14 min −1 ), suggesting substantially increased on and off rates for the ligand at mSUCNR1 compared to hSUCNR1. Importantly, when deriving the K d from the on and off rates for mSUCNR1, a value was obtained (1500 nM) that was still in close alignment with the K d measured in the saturation binding assays ( Figure 4B). To further explore the differences in binding kinetics of 22 between hSUCNR1 and mSUCNR1, residence times were calculated as 71 and 7.7 min for the two orthologs, respectively. As it was surprising to see such a large difference in residence times between the two species orthologs, we independently confirmed these results in more traditional dissociation rate experiments ( Figure S2C,D), which yielded k off rates of 0.057 min −1 and 0.32 min −1 for hSUCNR1 and mSUCNR1, respectively, both in good agreement with the values derived from our multiple concentration association kinetic studies.
Having now identified a fluorescent SUCNR1 agonist tracer, we next set out to develop a comparable antagonist tracer compound. Besides being complementary tools, antagonist tracers have a number of advantages over agonist tracers; e.g., they do not cause receptor desensitization or internalization. Since all current SUCNR1 antagonists are human specific, we focused our optimization only on hSUCNR1. First, we investigated if pre-arranging the biphenyl core of PB-20-OV24 (32) in a twisted conformation would improve the potency. We explored addition of methyl groups to three different orthopositions of 32 (Table 3). Having a methyl in the R 1 -position (33) led to more than a 2-fold decrease in potency, indicating space limitations in this direction in the binding pocket, while moving the methyl to the R 2 -position (34) increased the potency 3-fold. Adding the methyl to the second ring in the R 3position (35) proved the most successful and increased the potency almost 15-fold, thereby confirming our hypothesis. Next, alkoxyamines were explored, similar to the agonist series; however, surprisingly, compounds with NHBoc, represented here by 38, showed low-potency agonist activity rather than antagonism, and we therefore decided to explore the unprotected alkoxyamines instead. On these compounds, the R 3 -methyl surprisingly had the opposite effect on the longer C6chain, leaving the non-methylated analogue 39 slightly more potent than 40. However, decreasing the linker length to C5 (41) and C3 (42) increased the potency of the R 3 -methylated scaffold, almost regaining the potency of the chlorinated analogue 35. Since having a free amine seemed to be essential for having antagonistic activity on these extended compounds, we turned our attention back to the published antagonist 1 to investigate if the piperazine could be extended with a small linker for attachment of the fluorophore and in that way keep the piperazine amines to maintain antagonistic behavior. Indeed, this was possible, and extension with tert-butyl methylcarbamate (43) preserved the activity. We also wanted to see if addition of the R 3 -methyl to 1 would affect the potency and found a 4-fold increase in potency for 44, while the extended analogue 45 was only slightly more potent than the corresponding nonmethylated analogue 43. Finally, 45 was selected as the best tracer precursor, and introduction of NBD (46) yielded a tracer with potency comparable to that of the precursor.
We next aimed to confirm that 46 retained activity at the Nluc-hSUCNR1 receptor that would be used for the BRET binding assay, observing a pIC 50 of 6.75 ± 0.04 ( Figure 5A). In these experiments it was noted that, at high (10 μM) concentrations, 46 tended to not fully inhibit the cAMP response; however, 46 is a highly colored fluorescent compound, and it is likely that this is an artifact resulting from these properties. Saturation BRET binding experiments with 46 demonstrated clear specific binding to hSUCNR1, with a K d of 310 nM ( Figure 5B, Figure S3A). Not surprisingly, given the lack of ability of 46 as well as all other SUCNR1 antagonists we have tested to antagonize mSUCNR1 in cAMP assays, when tested in a saturation binding assay at mSUCNR1, 46 showed almost no specific binding ( Figure 5C). Kinetic analysis of 46 binding using hSUCNR1 produced k on = 61800 M −1 min −1 and k off = 0.035 min −1 , yielding an estimated K d value of 560 nM ( Figure  5D), in nice alignment with the saturation binding data ( Figure  5A). This off rate was independently verified using a traditional dissociation kinetic experiment, yielding a highly comparable K off value of 0.034 min −1 ( Figure S3B). Together, these data suggest that 46 is a useful antagonist fluorescent tracer for hSUCNR1 but not for mSUCNR1.
Next, we aimed to use 46 to understand why the antagonists are potent at hSUCNR1 but inactive at mSUCNR1. Previous work has identified two humanizing mutations in the rat (r) ortholog of SUCNR1, K18 1.31 E and K269 7.32 N, that are required to gain antagonist binding to rSUCNR1. 27 In mSUCNR1, these residues are N18 1.31 and K269 7.32 ; we therefore generated an N18 1.31 E/K269 7.32 N mSUCNR1 construct to establish if these mutations restore antagonist function. To avoid the need to make a stable cell line expressing this mutant receptor, we used a BRET-based assay that measures direct activation of Gα i2 36 in transiently transfected HEK-293T cells. Importantly, we first demonstrated that the mSUCNR1-N18 1.31 E/K269 7.32 N mutant retains the ability to respond to succinate in our G protein activation assay ( Figure S4A). Interestingly, unlike in our cAMP assay, in the G protein activation assay we observe somewhat higher potency for succinate at mSUCNR1 (pEC 50 = 5.29 ± 0.12) than hSUCNR1 (pEC 50 = 4.45 ± 0.23), consistent with previous reports that succinate is more potent at mSUCNR1 than hSUCNR1. 13 The pEC 50 of succinate at mSUCNR1-N18 1.31 E/K269 7.32 N in this assay was 4.76 ± 0.11, suggesting that these mutations may in part account for differences in succinate potency between human and mouse SUCNR1. However, when tested for antagonism against an EC 80 concentration of succinate, 1 still showed no measurable antagonism against mSUCNR1-N18 1.31 E/K269 7.32 N ( Figure  S4B).
This led us to further explore differences between rat, mouse, and human SUCNR1 to determine why mutation of these sites is sufficient for antagonist activity at rSUCNR1 but not mSUCNR1. Critically, when examining the residues shown to be involved in antagonist binding in the SUCNR1 crystal structure, 27 only one residue was identified that is conserved in human (W88 EL1 ) and rat (W84 EL1 ) but not mouse (G84 EL1 ) SUCNR1 ( Figure 6A). We therefore generated an additional mutant mSUCNR1 construct containing N18 1.31 E, K269 7.32 N, and G84 EL1 W and first demonstrated in our G protein activation assay that this mutant is activated by succinate with a pEC 50 of 4.28 ± 0.09 ( Figure S4A). Satisfyingly, when we tested 1 as an antagonist against an EC 80 concentration of succinate at this mutant, a level of antagonism similar to that with hSUCNR1 was observed ( Figure S4B). Together, this suggests that these three residues are critical for the lack of activity of antagonists at mSUCNR1 and that incorporation of N18 1.31 E, K269 7.32 N, and G84 EL1 W mutations is sufficient to produce a humanized (hm) version of mSUCNR1. We therefore established a hmSUCNR1 cell line and used this to confirm that 1 potently inhibits hmSUCNR1 in our cAMP assay ( Figure 6B). Indeed, in line with what was observed in the G protein activation assay, 1 inhibited succinate response at hmSUCNR1 to a similar degree to how it inhibits hSUCNR1, with a pIC 50 of 7.10 ± 0.10. Having demonstrated functional antagonism at hmSUCNR1, we used our fluorescent tracer compound to assess antagonist binding to hmSUCNR1. In line with our cAMP results, clear specific binding was observed for the tracer antagonist, 46, with a K d of 390 nM ( Figure 6C, Figure S5A). This affinity is very similar to the K d obtained for 46 at hSUCNR1 (310 nM), suggesting that these three residues fully account for the lack of antagonist binding to mSUCNR1. In addition to binding the tracer antagonist, hmSUCNR1 also broadly maintains agonist binding, as both agonist tracers, 22 (K d = 490 nM; Figure 6D, Figure S5B) and 7 (K d = 2650 nM; Figure S5C) bind effectively to hmSUCNR1.
Having identified a tracer agonist for human and mouse SUCNR1 and a tracer antagonist for human and humanized mouse SUCNR1, we set out to use these compounds to assess binding affinity of various unlabeled SUCNR1 ligands. Ten SUCNR1 ligands were chosen and tested in competition binding assays using concentrations of 22 tracer comparable to its calculated K d at hSUCNR1 (3 μM), mSUCNR1 (1 μM), or hmSUCNR1 (1 μM) (Table 4), including previously published cis-epoxysuccinic acid (47) and an example antagonist from the patent literature 37 (48). Across the testing we consistently observed that all agonists displayed comparable binding to all three SUCNR1 receptors ( Figure 7A−C). However, as predicted, most antagonists tested bound with high affinity only to hSUCNR1 and hmSUCNR1 ( Figure 7D−F). Next, to compare the competition binding data obtained using our agonist vs antagonist tracer, we tested the same set of compounds in competition binding assays using 46 as the tracer (562 nM) at hSUCNR1 and hmSUCNR1 (Table 4). An excellent correlation was observed between the pK i values obtained using the two different fluorescent ligands at both hSUCNR1 ( Figure 7G; r = 0.98) and hmSUCNR1 ( Figure 7H; r = 0.98), confirming that both the agonist and antagonist tracer ligands are binding competitively to the same site of SUCNR1. In addition, the affinities across all compounds tested at hmSUCNR1 correlated much better with the affinities for these compounds at hSUCNR1 (r = 0.94) than the affinities obtained for these compounds at mSUCNR1 ( Figure 7I; r = 0.39) Competion binding using 46 a,c hSUCNR1 pK i mSUCNR1 pK i hmSUCNR1 pK i hSUCNR1 pK i hmSUCNR1 pK i These competition binding studies established pK i affinity values for succinate ranging from 3.0 to 3.7 across the three different receptor constructs. These data suggest a relatively low affinity of succinate for SUCNR1, suggesting that functional succinate responses rely on strong signal amplification and a receptor reserve to produce the higher pEC 50 potencies (∼5.0) observed at both hSUCNR1 and mSUCNR1 in the cAMP assay. The same was true for the other agonists tested, for example 9d, where the measured affinity (pK i = 6.2−6.3) was notably lower than the potency for this compound in cAMP assays (pEC 50 = 7.21−7.44). In contrast, antagonist affinities were comparable to, or slightly higher than, the IC 50 values obtained in cAMP assays. For example, 1 yielded pK i values between 7.10 and 7.4 for hSUCNR1, compared with an pIC 50 value of 7.07 in the cAMP assay.
One interesting observation from our competition studies using hmSUCNR1 is that these humanizing mutations have not had the same effect across all antagonists. Of particular note, while antagonists related to 1 have activity completely restored in hmSUCNR1, 32 and 35 that lack the piperazine group show only a partial recovery of function, with the affinity remaining ∼10-fold lower at hmSUCNR1 compared to hSUCNR1. This cannot be explained by docking of 32 in homology models of hor hmSUCNR1 where all residues in the binding pocket are conserved and the binding poses are nearly identical. Thus, differences outside the binding pocket must be responsible for the observed difference in affinity for the smaller antagonists at human and humanized mouse SUCNR1, and the ionic interaction between the piperazine and Glu18 on the larger antagonists fully compensates for this difference.
The patent derived antagonist 48, which represents a different chemical scaffold, was found to be human specific and did not regain any activity on hmSUCNR1, but it could displace both the agonist and antagonist tracer, demonstrating that the compound binds to the same site as the others.

■ CONCLUSIONS
We have developed the first potent fluorescent tracer agonist for SUCNR1, 22. We have demonstrated that this tracer ligand can be used to measure both binding affinity and binding kinetics to SUCNR1 in living cells using a BRET-based assay. Interestingly, although our best tracer agonist, 22, binds with comparable affinity to the human and mouse receptor orthologs, it does so with significantly different kinetic properties. Specifically, 22 has much faster association and dissociation rates, as well as a shorter residence time at mSUCNR1 compared to hSUCNR1. In addition, we have also developed the first fluorescent SUCNR1 antagonist, 46, and used this ligand to demonstrate that three residues that differ between human and mouse SUCNR1 account for the lack of binding of currently known SUCNR1 antagonists to the mouse receptor. We further use both 22 and 46 in a range of competition binding studies to demonstrate that both ligands bind to the same site on SUCNR1. Together, these fluorescent tracers provide a new way to measure binding to SUCNR1 in intact living cells and are expected to be invaluable tools in drug discovery efforts targeting this receptor. ■ EXPERIMENTAL SECTION General Remarks. All commercially available starting materials and solvents were used without further purification unless otherwise stated. DCM, THF, and DMF were dried using a Glass Contour Solvent System built by SG Water USA. MeCN and NEt 3 were distilled over CaH 2 and stored under activated 4 Å molecular sieves; MeOH and N,N-diisopropylethylamine were dried over 3 Å molecular sieves. TLC was performed on TLC silica gel 60 F254 plates and visualized at 254 and/or 365 nm. Purification by flash chromatography was carried out using silica gel 60 (0.040−0.063 mm, Merck). Purification by automated flash chromatography was performed on a RevelerisX2 Flash Chromatography System, Buchi. 1 H and 13 C spectra were recorded at 400 and 600 MHz, 101 and 151 MHz, respectively, on Bruker Avance III (400 MHz) or Bruker Avance III HD (600 MHz) instruments at 300 K and calibrated relative to residual solvent peaks. HPLC analysis was performed on an UltiMate HPLC system (Thermo Scientific) using a Gemini-NX C18 column (3 μm 1) over 5 min, flow rate 1 mL/min. High-resolution mass spectra (HRMS) were recorded on a QExactive Orbitrap mass spectrometer equipped with a SMALDI5 ion source. The sample was analyzed in the positive ion mode using a peak from the DHB matrix for internal mass calibration whereby a mass accuracy of 2 ppm or better was achieved. Purity was determined by HPLC and confirmed by inspection of NMR spectra. The purity of all test compounds was >95%.
5-((tert-Butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate: tert-Butyl (5-hydroxypentyl)carbamate was dissolved in DCM (11 mL) in a flask under an argon atmosphere. The mixture was cooled on an ice−water bath before addition of tosyl chloride (1217 mg, 6.38 mmol) followed by pyridine (0.85 mL, 10.49 mmol). The reaction mixture was stirred at rt for 21 h. After completion, the reaction mixture was washed with aqueous 1 M HCl (×2). The aqueous phases were combined and re-extracted with DCM (×2). The organic phases were combined, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated in vacuo. . Spectra in accordance with reported data. 38 5-((tert-Butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate (32 mg, 0.09 mmol) was dissolved in MeCN (0.6 mL) in a dry flask under an argon atmosphere. Then, 4 2 (20 mg, 0.06 mmol), K 2 CO 3 (17 mg, 0.12 mmol), and MeCN (0.6 mL) were added to the flask. The reaction mixture was stirred at 80°C for 2 days. After completion, water was added to the reaction mixture. The aqueous phase was extracted with EtOAc (×3). The organic phases were combined, washed with brine, dried over MgSO 4 , filtered, and concentrated in vacuo. give the product as an amber oil that was used directly in the next step (R f = 0.66 (10% MeOH in DCM)).
BRET Binding Assays. BRET binding assays were carried out using Flp-In T-REx 293 cells engineered to inducibly express human or mouse SUCNR1 tagged at their N terminal with Nluc. To help ensure proper membrane expression of the Nluc-SUCNR1 constructs, the signal peptide sequence for the mGlu5 glutamate receptor was added to the construct immediately before the Nluc sequence. For binding assays, cells were plated in poly-D-lysine-coated 96-well plates (white or black with clear bottom), and Nluc-SUCNR1 expression was induced with overnight Dox treatment (100 ng/mL). Prior to the assays, culture medium was removed, and cells were washed twice with HBSS. Cells were then incubated in HBSS at 37°C for 30 min. The NanoGlo Nluc substrate (Promega, N1110) was then added to the cells, at a final 1:800 dilution, and incubated for 10 min. For equilibrium assays (saturation or competition), the indicated fluorescent or non-fluorescent compounds were added, and plates were incubated for a further 5 min at 37°C before reading. Luminescent emissions at 545 and 460 nm were measured using a ClarioStar microplate reader (BMG Labtech). For kinetic assays, luminescent emissions at 535 and 475 nm were measured using a PheraStar plate reader (BMG Labtech) at the indicated time intervals. For association kinetics, baseline readings were taken before the addition of test compounds, followed by injection of the test compound by the plate reader. For dissociation kinetics, baseline measurements were taken for 2 min with cells incubated with the indicated concentration of tracer ligand, followed by injection of a 100 μM concentration of the competing ligand by the plate reader to measure dissociation of the tracer. BRET ratios were calculated as 545/ 460 (ClarioStar assays) or 535/475 (PheraStar assays).
G Protein Activation BRET Assay. G protein activation assays were carried out using the open-source TRUPATH platform previously described. 36 HEK-293T cells were transfected with Gαi1-Rluc8, Gγ8-GFP 2 , Gβ3 and either human, mouse, mouse-N18E/K269N, or mouse-N18E/G84W/K269N SUCNR1 in a 1:1:1:1 plasmid mass ratio using polyethyleneimine. After 24 h, cells were trypsinized and seeded into poly-D-lysine-coated white 96-well plates. After a further 24 h in culture, medium was removed, cells were washed twice with HBSS and incubated in HBSS at 37°C for 30 min. Prolume purple substrate (NanoLight Technology, Cat. No. 369) was added to the cells at a final 5 μM concentration and incubated for 10 min at 37°C. A background measurement was then taken by reading luminescent emission at 525 and 385 nm using a ClarioStar microplate reader (BMG Labtech). Succinate was added to the cells and incubated for a further 5 min at 37°C before reading luminescence again at 525 and 385 nm again. For the antagonist assays, 1 was added to the cells and incubated for 5 min before addition of prolume purple and a total of 15 min before succinate was added. For analysis, the ratio of 525/385 emission was taken and expressed as a fold change before and after the addition of succinate.
Computational Modeling. The X-ray structure of the humanized rSUCNR1 (PDB code 6RNK) 27 was cleaned from excess water and other additives and prepared using Protein Preparation in Maestro (Schrodinger, LCC, version 12.7.161). Three mutations, E18N, N269K, and W84G, were introduced to make the murinized rSUCNR1 model. 9d and 1 were prepared using ligprep (OPLS4 forcefield and standard settings) and docked in the humanized and murinized rSUCNR1 using InducedFit docking with Glide Extra Precision (XP) and default settings with the box centered around 1.